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Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA
1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Abstract:
Protein F is a fibronectin-binding surface protein of Streptococcus pyogenes (group A streptococcus) that mediates adherence to host cells. A gene product encoded by rofA activates transcription of the gene that encodes protein F (prtF) and was identified in a strain of S. pyogenes that expressed high levels of protein F under all conditions tested. Insertional inactivation of rofA in this strain results in a phenotype similar to that of other strains where high-level transcription of prtF occurs only in response to increased oxygen tension. In this study, we have compared the regulation of prtF and rofA in O2-regulated and constitutive strains in order to gain further insight into the function of rofA. Comparison of the prtF and rofA transcripts by S1 nuclease and primer extension assays indicated that the same promoters for each transcript are used in both O2-regulated and constitutive strains. However, analyses of rofA-lacZ reporter alleles revealed that a key difference between strains involves regulation of rofA itself. In O2-regulated strains, expression of rofA was elevated following culture under conditions of reduced O2 tension. However, a much more robust activation of rofA expression was observed when constitutive strains were grown under similar conditions. Exchange of reporter and rofA alleles between strains demonstrated that host genetic background, and not the sequence of the respective rofA allele or regulatory region, dictates the expression phenotype. Activation of rofA required RofA, and RofA was shown to bind specifically to DNA containing the promoters for rofA and prtF. Finally, overexpression of either allele of rofA caused constitutive expression of prtF regardless of host background. These data suggest a model where anaerobic expression of prtF in constitutive hosts is controlled at the level of transcription of rofA and implicate additional factors in this regulatory pathway.
Insights
Regulation of Streptococcus pyogenes protein F (prtF) involves the rofA gene. This study reveals that host genetic background, not rofA sequence, dictates expression, with RofA protein binding to prtF and rofA promoters.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus pyogenes protein F (prtF) mediates host cell adherence.
- The rofA gene product regulates prtF transcription, influencing protein F expression.
- Strain variations exist in prtF regulation, with some strains showing constitutive expression and others oxygen-dependent regulation.
Purpose of the Study:
- To investigate the regulatory mechanisms of prtF and rofA in oxygen-regulated and constitutive Streptococcus pyogenes strains.
- To elucidate the role of the rofA gene product in controlling prtF expression.
- To understand the genetic basis for differential regulation of prtF.
Main Methods:
- S1 nuclease and primer extension assays to compare prtF and rofA transcripts.
- Analysis of rofA-lacZ reporter alleles to assess gene expression.
- Allelic exchange experiments to determine the influence of host genetic background.
- DNA binding assays to confirm RofA-promoter interactions.
Main Results:
- Identical promoters for prtF and rofA are used in both oxygen-regulated and constitutive strains.
- Regulation of rofA itself differs between strains; expression is induced by low oxygen, with stronger activation in constitutive strains.
- Host genetic background, not rofA allele sequence, determines the expression phenotype.
- RofA protein binds specifically to the promoters of both rofA and prtF.
- Overexpression of rofA leads to constitutive prtF expression irrespective of host background.
Conclusions:
- Anaerobic expression of prtF in constitutive hosts is primarily controlled by the transcriptional regulation of rofA.
- Host genetic factors play a crucial role in determining rofA and prtF expression patterns.
- Additional regulatory factors likely participate in the control of prtF expression under anaerobic conditions.